In conclusion, we provide for the first time that gefitinib can induce cancer cell apoptosis through an EGFR-independent pathway. Understanding the mechanisms by which securin and ATF3 signal transduction pathways regulate apoptosis following treatment with gefitinib may contribute to the novel therapeutic strategies in cancers.
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Gefitinib (μM, 24 h)
Fig. 1. The effect of gefitinib on the cell viability in a variety of human cancer cell lines. The cells were treated with 0-60 μM gefitinib for 24 h. After drug
treatment, the cells were washed with PBS and incubated for 2 days. The cell viability was measured by MTT assay. The results were obtained from four to seven experiments and the bar represents the mean ± S.E.M. *p < 0.05 and **p
< 0.01 indicate significant difference between control and gefitinib treated samples.
Fig. 2. The protein level of phospho-EGFR and total EGFR in a variety of human cancer cell lines. The total protein extracts from various cancer cell lines including RKO, A549, BFTC905, MCF7 and A375 were subjected to western blot analysis using specific anti-phospho-EGFR, anti-EGFR and anti-actin antibodies. The representative western blot data were shown from one of three separate experiments with similar findings. Actin was a loading control. Positive
control of phospo-EGFR and EGFR were extracted proteins from A431 cells.
(A)
Fig. 3. The effect of gefitinib on the cell cycle progression and sub-G1 formation in A375 and BFTC905 cells. (A) The cells were treated with 0-60 μM gefitinib for
(A)
Fig. 4. The effect of gefitinib on apoptosis in A375 and BFTC905 cells. (A) After .treatment with or without gefitinib for 24 h, the cells collected and then .subjected to annexin V and PI staining. The representative flow data were .shown from one of three to four separate experiments with similar findings. The .results represented the mean ± S.E.M. (B) The percentage of apoptosis .populations (early and late stages) were quantified by CellQuest software. The .results were obtained from three to four experiments and the bar represents the .mean ± S.E.M. **p < 0.01 indicates significant difference between control and
Annexin V integral
Fig. 5. The time-lapse observation of apoptosis by gefitinib. A375 cells were plated at a density of 2 × 105 cells/p35 Petri dish for 24 h, then the cells were treated with or without 60 μM gefitinib for 24 h. The cell morphology was observed under a living cell imaging system. The representative cell morphology data were shown from one of three separate experiments with similar findings. The
arrows indicated the apoptotic cells.
0 h 8 h 16 h 24 h
A375 cells (gefitinib 60 μM)
0 h 8 h 16 h 24 h
A375 cells (control)
10 μm
10 μm
(A)
(B)
A375 cells
Gefitinib (μM, 24 h)
0 20 40
Mitochondrial membrance potential (intensity of DiOC6)
0
Fig. 6. The effect of gefitinib on the hyperpolarization of mitochondria. (A) After .treatment with or without gefitinib for 24 h, the cells were incubated with 50 .nM DiOC6 and then analyzed by a flow cytometer. The representative flow .data were shown from one of five separate experiments with similar findings.
.(B) The fluorescence intensity of DiOC6 were quantified by CellQuest software.
.The results were obtained from five experiments and the bar represents the .mean ± S.E.M. *p < 0.05 and **p < 0.01 indicate significant difference between
Cell counts
Fig. 7. The effect of gefitinib on the activation of caspase-3 and the cleavage of
(A)
(B)
A375 cells
Gefitinib (μM, 24 h)
0 10 20 40 60
Relative protein level of securin (fold) 0.2
0.4
Fig. 8. The effect of gefitinib on the securin protein expression. (A) A375 cells were treated with 0-60 μM gefitinib for 24 h. The total protein extracts were prepared for immunoblot analysis using anti-securin and anti-actin antibodies. The representative western blot data were shown from one of three separate experiments with similar findings. Actin was a loading control. (B) The relative protein level of securin were from western blot by semi-quantification. The results were obtained from three experiments and the bar represents the mean ± S.E.M. *p < 0.05 and **p < 0.01 indicate significant difference between control and gefitinib treated samples.
Fig. 9. The effect of gefitinib on the expression and location of securin. A375 cells were treated with or without 40 μM gefitinib for 24 h. At the end of treatment, the cells were incubated with mouse anti-securin antibody and then incubated with goat anti-mouse FITC. The β-tubulin and nuclei were stained with the Cy3- labeled anti-β-tubulin and Hoechst 33258, respectively.
control
gefitinib (40 μM, 24 h)
control
gefitinib (40 μM, 24 h)
nuclei β-tubulin securin merge
A375 cells
100 μm
100 μm
20 μm
20 μm
HCT116 cells
Gefitinib (μM, 24 h)
0 10 20 40 60
Cell viabilit y ( % )
10 20 30 40 50 6070 8090 100110 120
securin (+/+) securin (-/-)
**
**
** **
* *
#
Fig. 10. The comparison of cytotoxicity between the securin-wild type and -null .HCT116 cancer cells following gefitinib treatment. The cells were treated .with 0-60 μM gefitinib for 24 h. After drug treatment, the cells were washed .with PBS and incubated for 2 days. The cell viability was measured by MTT .assay. The results were obtained from six experiments and the bar represents .the mean ± S.E.M. *p < 0.05 and **p < 0.01 indicate significant difference .between control and gefitinib treated samples. #p < 0.05 indicates significant .difference between the securin-wild type and -null HCT116 cancer cells by the .gefitinib treatment at the same concentration.
A375 cells
Fig. 11. The effect of the overexpression of securin on the gefitinib-induced cytotoxicity. The cells were transfected the control or securin vectors and
(A)
(B)
A375 cells
Gefitinib (μM, 24 h)
0 10 20 40 60
Relative protein level of ATF3 (fold) 0.5
1.0 1.5 2.0 2.5 3.0
*
**
Fig. 12. The effect of gefitinib on the ATF3 protein expression. (A) A375 cells were treated with 0-60 μM gefitinib for 24 h. The total protein extracts were prepared for immunoblot analysis using anti-ATF3 and anti-actin antibodies.
The representative western blot data were shown from one of three separate experiments with similar findings. Actin was a loading control. (B) The relative protein level of ATF3 were from western blot by semi-quantification.
The results were obtained from three experiments and the bar represents the mean ± S.E.M. *p < 0.05 and **p < 0.01 indicate significant difference
A375 cells Gefitinib
(μM, 24 h)
Actin ATF3
0 10 20 40 60
Fig. 13. The effect of gefitinib on the expression and location of ATF3. A375 cells were treated with or without 40 μM gefitinib for 24 h. At the end of treatment, the cells were incubated with rabbit anti-ATF3 antibody and then incubated with goat anti-rabbit Cy-3. The F-actin and nuclei were stained with BODIPY FL phallacidin and Hoechst 33258, respectively.
control
gefitinib (40 μM, 24 h)
control
gefitinib (40 μM, 24 h)
nuclei F-actin ATF3 merge
A375 cells
100 μm
100 μm
20 μm
20 μm
RKO cells
Fig. 14. The comparison of cytotoxicity between the gefitinib and its analogues in a variety of human cancer cell lines. The cells were treated with 0-80 μM gefitinib or its analogues for 24 h. After drug treatment, the cells were washed with PBS and incubated for 2 days. The cell viability was measured by MTT assay. The results were obtained from three to seven experiments and the bar represents the mean ± S.E.M. **p < 0.01 indicates significant difference
(A)
(B)
Fig. 15. The schematic diagram of antitumor activity by gefitinib
Fig. 16. A proposed model for the gefitinib-induced apoptosis